Limits...
Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation.

Todorovic V, Koetsier JL, Godsel LM, Green KJ - Mol. Biol. Cell (2014)

Bottom Line: Moreover, Pkp3 forms a complex with Rap1 GTPase, promoting its activation and facilitating desmosome assembly.We show further that Pkp3 deficiency causes disruption of an E-cadherin/Rap1 complex required for adherens junction sealing.These findings reveal Pkp3 as a coordinator of desmosome and adherens junction assembly and maturation through its functional association with Rap1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.

Show MeSH

Related in: MedlinePlus

Pkp3 is required for efficient assembly of DP into desmosomes. (A) Immunofluorescence staining for DP representing the distinct patterns of its disruption in Pkp2, Pkp3, and Pkp2-3 double-KD SCC9 cells. Scale bar, 20 μm. Yellow dashed rectangles in top images delineate areas enlarged at the bottom. Yellow arrows point to the sites of cell–cell contacts; red arrows point to DP-containing nonvesicular desmosome precursors in the cytoplasm. Note the absence of cytoplasmic particles in Pkp3 KD and both particles and cell–cell border DP in double-KD cells. (B) Average fluorescence DP pixel intensity at the cell–cell borders and inside the cytoplasm, measured for ≥100 individual cells, showed decreased DP at cell–cell borders in Pkp3 KD cells and a corresponding increase in the cytoplasm. Error bars represent ± SEM. ***p < 0.001 (ANOVA, Bonferroni). (C) Western blot showing the efficiency of Pkp3 siRNA KD in 3D organotypic raft after 6 d of culture. (D) Representative immunofluorescence images of 3D raft cultures after 6 d of differentiation show diffuse cytoplasmic DP distribution in Pkp3-ablated rafts. Scale bar, 50 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4230782&req=5

Figure 2: Pkp3 is required for efficient assembly of DP into desmosomes. (A) Immunofluorescence staining for DP representing the distinct patterns of its disruption in Pkp2, Pkp3, and Pkp2-3 double-KD SCC9 cells. Scale bar, 20 μm. Yellow dashed rectangles in top images delineate areas enlarged at the bottom. Yellow arrows point to the sites of cell–cell contacts; red arrows point to DP-containing nonvesicular desmosome precursors in the cytoplasm. Note the absence of cytoplasmic particles in Pkp3 KD and both particles and cell–cell border DP in double-KD cells. (B) Average fluorescence DP pixel intensity at the cell–cell borders and inside the cytoplasm, measured for ≥100 individual cells, showed decreased DP at cell–cell borders in Pkp3 KD cells and a corresponding increase in the cytoplasm. Error bars represent ± SEM. ***p < 0.001 (ANOVA, Bonferroni). (C) Western blot showing the efficiency of Pkp3 siRNA KD in 3D organotypic raft after 6 d of culture. (D) Representative immunofluorescence images of 3D raft cultures after 6 d of differentiation show diffuse cytoplasmic DP distribution in Pkp3-ablated rafts. Scale bar, 50 μm.

Mentions: Because DP is an obligate desmosome component that serves as a general marker of junction assembly state, we next tested how ablating Pkp3 affects its cellular localization. In steady state, most DP is localized at cell–cell contacts (Figure 2A and Supplemental Figure S1, B, C, E, and F). Frequently, some unincorporated cytoplasmic particles can be observed in the cortical region adjacent to the plasma membrane, but these are fewer than what can be observed during de novo assembly of desmosomes. Pkp2 KD cells exhibit the previously reported “beads-on-a-string” appearance characterized by the alignment of cytoplasmic DP particles along intermediate filaments and reduced DP at cell–cell contacts (Bass-Zubek et al., 2008; Figure 2A, see lower high-magnification inset). In contrast to the “beads-on-a-string” appearance of Pkp2-ablated cells, Pkp3 KD SCC9 and HaCaT cells exhibited a diffuse cytoplasmic DP localization with large DP clusters at the sites of cell contact, albeit sparsely distributed along the border (Figure 2A and Supplemental Figure S1, C and E). Pkp2/3 double-KD cells exhibited only diffuse DP cytoplasmic staining, with no DP present at the cell borders (Figure 2A). The restricted distribution of DP along the plasma membrane in Pkp3 KD cells correlated with a significant decrease in average fluorescent intensity of DP at cell–cell interfaces. A concomitant increase in cytoplasmic intensity of DP was observed in Pkp3-silenced cells compared with control cells (Figure 2B). This effect of Pkp3 ablation on DP redistribution from the cell–cell borders to a more diffuse cytoplasmic distribution is preserved in a three-dimensional (3D) organotypic model that recapitulates the differentiation and morphogenesis program exhibited by developing epidermis (Figure 2, C and D). In contrast to Pkp3- mice (Sklyarova et al., 2008), Pkp3-silenced human raft cultures do not exhibit compensatory overexpression of Pkp1, and the presence of Pkp1 (Supplemental Figure S1G) does not seem to be able to compensate for Pkp3 loss in providing for proper DP localization at the sites of cell–cell contact.


Plakophilin 3 mediates Rap1-dependent desmosome assembly and adherens junction maturation.

Todorovic V, Koetsier JL, Godsel LM, Green KJ - Mol. Biol. Cell (2014)

Pkp3 is required for efficient assembly of DP into desmosomes. (A) Immunofluorescence staining for DP representing the distinct patterns of its disruption in Pkp2, Pkp3, and Pkp2-3 double-KD SCC9 cells. Scale bar, 20 μm. Yellow dashed rectangles in top images delineate areas enlarged at the bottom. Yellow arrows point to the sites of cell–cell contacts; red arrows point to DP-containing nonvesicular desmosome precursors in the cytoplasm. Note the absence of cytoplasmic particles in Pkp3 KD and both particles and cell–cell border DP in double-KD cells. (B) Average fluorescence DP pixel intensity at the cell–cell borders and inside the cytoplasm, measured for ≥100 individual cells, showed decreased DP at cell–cell borders in Pkp3 KD cells and a corresponding increase in the cytoplasm. Error bars represent ± SEM. ***p < 0.001 (ANOVA, Bonferroni). (C) Western blot showing the efficiency of Pkp3 siRNA KD in 3D organotypic raft after 6 d of culture. (D) Representative immunofluorescence images of 3D raft cultures after 6 d of differentiation show diffuse cytoplasmic DP distribution in Pkp3-ablated rafts. Scale bar, 50 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4230782&req=5

Figure 2: Pkp3 is required for efficient assembly of DP into desmosomes. (A) Immunofluorescence staining for DP representing the distinct patterns of its disruption in Pkp2, Pkp3, and Pkp2-3 double-KD SCC9 cells. Scale bar, 20 μm. Yellow dashed rectangles in top images delineate areas enlarged at the bottom. Yellow arrows point to the sites of cell–cell contacts; red arrows point to DP-containing nonvesicular desmosome precursors in the cytoplasm. Note the absence of cytoplasmic particles in Pkp3 KD and both particles and cell–cell border DP in double-KD cells. (B) Average fluorescence DP pixel intensity at the cell–cell borders and inside the cytoplasm, measured for ≥100 individual cells, showed decreased DP at cell–cell borders in Pkp3 KD cells and a corresponding increase in the cytoplasm. Error bars represent ± SEM. ***p < 0.001 (ANOVA, Bonferroni). (C) Western blot showing the efficiency of Pkp3 siRNA KD in 3D organotypic raft after 6 d of culture. (D) Representative immunofluorescence images of 3D raft cultures after 6 d of differentiation show diffuse cytoplasmic DP distribution in Pkp3-ablated rafts. Scale bar, 50 μm.
Mentions: Because DP is an obligate desmosome component that serves as a general marker of junction assembly state, we next tested how ablating Pkp3 affects its cellular localization. In steady state, most DP is localized at cell–cell contacts (Figure 2A and Supplemental Figure S1, B, C, E, and F). Frequently, some unincorporated cytoplasmic particles can be observed in the cortical region adjacent to the plasma membrane, but these are fewer than what can be observed during de novo assembly of desmosomes. Pkp2 KD cells exhibit the previously reported “beads-on-a-string” appearance characterized by the alignment of cytoplasmic DP particles along intermediate filaments and reduced DP at cell–cell contacts (Bass-Zubek et al., 2008; Figure 2A, see lower high-magnification inset). In contrast to the “beads-on-a-string” appearance of Pkp2-ablated cells, Pkp3 KD SCC9 and HaCaT cells exhibited a diffuse cytoplasmic DP localization with large DP clusters at the sites of cell contact, albeit sparsely distributed along the border (Figure 2A and Supplemental Figure S1, C and E). Pkp2/3 double-KD cells exhibited only diffuse DP cytoplasmic staining, with no DP present at the cell borders (Figure 2A). The restricted distribution of DP along the plasma membrane in Pkp3 KD cells correlated with a significant decrease in average fluorescent intensity of DP at cell–cell interfaces. A concomitant increase in cytoplasmic intensity of DP was observed in Pkp3-silenced cells compared with control cells (Figure 2B). This effect of Pkp3 ablation on DP redistribution from the cell–cell borders to a more diffuse cytoplasmic distribution is preserved in a three-dimensional (3D) organotypic model that recapitulates the differentiation and morphogenesis program exhibited by developing epidermis (Figure 2, C and D). In contrast to Pkp3- mice (Sklyarova et al., 2008), Pkp3-silenced human raft cultures do not exhibit compensatory overexpression of Pkp1, and the presence of Pkp1 (Supplemental Figure S1G) does not seem to be able to compensate for Pkp3 loss in providing for proper DP localization at the sites of cell–cell contact.

Bottom Line: Moreover, Pkp3 forms a complex with Rap1 GTPase, promoting its activation and facilitating desmosome assembly.We show further that Pkp3 deficiency causes disruption of an E-cadherin/Rap1 complex required for adherens junction sealing.These findings reveal Pkp3 as a coordinator of desmosome and adherens junction assembly and maturation through its functional association with Rap1.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611.

Show MeSH
Related in: MedlinePlus